I propose to study the molecular mechanisms of transport of organic substrates in specific renal cell types. Na-dependent transport systems in proximal tubular cells represent the dominant pathways for reabsorption in the kidney, yet the mechanism(s) for this type of 'coupled'-transport remains to be elucidated for any class of substrates. Such information is necessary to understand basic questions concerning the movement of molecules across cell membranes. The proposed approach involves measuring transport in three systems: intact, isolated cells, ATP-depleted cell 'models', and apical (brush border) membrane vesicles. Each system offers unique advantages in characterizing transport in the kidney. In particular I propose to study the transport of four different subtrates: D-glucose, L-lactate, L-proline, and succinate. Each is a model substrate for a separate Na-dependent pathway, and each represents an important class of compounds in renal metabolism. I will examine the effect of membrane potential and ionic composition on the kinetics of transport of these compounds, and determine the effects on transport of various classes of inhibitors. Basal-lateral transport processes will be examined by comparing transport of each substrate in both brush border membrane vesicles and in intact, isolated cells under similar expermental conditions. The data gained from these studies will be used to develop models describing the qualitative and quantitative characteristics of transport of each class of test substrates. Long term goals include the preparation of purified distal tubular cells and the separation of proximal cells into enriched populations of S1 and S2 cells. These procedures will permit the characterization of transport processes in each cell type, and will provide systems for biochemical and tissue culture studies.